Guía de Referencia para la Certificación de Sistemas Inteligentes de Transporte (ITS)

3.3.4.1 Endothelial function: Flow mediated dilation (FMD)

Determination of the presence of endothelial dysfunction at the systemic level in

neurodegenerative disease could provide important information about the involvement of macro-vascular function in the pathogenesis of the disease.

FMD is considered the gold standard technique for assessing systemic endothelial function 199. It is a well-established technique that has been widely used in clinical research to assess peripheral vascular function and cardiovascular disease risk factors

700, 701_{. Its main advantage over other techniques is that it is non-invasive and by nature}

of this, allows repeated measurements to be taken either sequentially or over time with a high level of patient acceptance 52. Its only disadvantage is that it requires practice to master the technique and carefully controlled experimental conditions are required to ensure reproducibility and reliability of results 52, 702.

FMD refers to the dilation of a vessel in response to increased blood flow. A sudden increase in blood flow is known to exert a shear stress stimulus on the vessel wall which, under normal circumstances, triggers the release of the vasodilator NO from the endothelium leading to an increase in vessel diameter to accommodate the increased blood flow. A failure to observe this vascular dilation response to increased flow is considered indicative of endothelial dysfunction and hence important information about the functioning of the endothelium can be gained by assessing the dilation response of a systemic artery to an experimentally produced increase in blood flow in FMD.

3.3.4.2 Protocol

Measurement of endothelium dependent FMD in this thesis has been conducted in accordance with the guidelines for assessment published by Corretti et al 702.

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The procedure involves ultrasound imaging of the brachial artery of the upper arm, with an ultrasound probe positioned in the longitudinal plane, 5-10cm above the antecubital fossa, using a 2D colour Doppler ultrasound system (CDI).

Prior to all FMD assessments conducted in this thesis, patients were required to undergo a 12 hour fast due to the potential influence that food intake and caffeine can have on flow mediated vascular reactivity 702. Furthermore, as temperature can also influence vascular reactivity all procedures were conducted in a quiet, dark, temperature controlled room at 22OC. The measurement procedure is summarised as follows:

• The patient is positioned supine and allowed to rest in this position for 10 minutes prior to first scan.

• Their arm is then positioned comfortably in an extended position and an ultrasound image of the brachial artery is obtained from the upper right arm • A segment of the artery with clear anterior and posterior intimal-lumen interfaces

is selected on the imaging screen to ensure optimum recording of vessel diameter changes (figure 3.7)

Figure 3.7 Imaging screen in FMD with brachial artery segment highlighted

• 2 minutes of baseline vessel diameter readings are taken from the brachial artery • A 5.6 inch wide BP cuff (sphygmomanometer) positioned at the forearm is then

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minutes. The inflated cuff effectively occludes the blood flow through the brachial artery, inducing hypoxia and causing dilation of downstream resistance vessels. • After 5 minutes the cuff is rapidly deflated and the brachial artery is imaged for a

further 2 minutes to assess the hyperaemic response. (figure 3.8)

Figure 3.8 Breakdown of the 7 minute FMD examination protocol

On deflation of the cuff, the brachial artery is no longer occluded and there is a sudden increase of blood flow through it (hyperaemia). As mentioned previously this increased blood flow produces a shear stress stimulus on the vessel wall which should trigger the endothelium to release NO, possibly in combination with other vasodilators, and an increase in vessel diameter should be seen. A failure to observe a good vasodilation response would indicate systemic endothelial dysfunction.

Following FMD assessment and after a 10 minute rest period, the procedure is repeated using a sublingual tablet of nitroglycerin (GTN 0.3mg) in place of the

sphygmomanometer. GTN is converted by the body into NO and this then acts directly on the vascular smooth muscle cells to bring about vasodilation, independent of the endothelium. GTN is therefore used to confirm whether any impaired dilation response detected by FMD can definitely be attributed to a dysfunctional endothelium and is not simply the result of a vascular smooth muscle dysfunction. It would be expected that, even in the presence of endothelial dysfunction, nitroglycerin mediated vasodilation (NMD) should be unchanged. If NMD were significantly impaired to the same degree as

0 2 5 7

Baseline Hyperaemia Cuff inflated Cuff deflated

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FMD it would suggest the vascular smooth muscle is not functioning properly and any impaired FMD responses could not be convincingly attributed to endothelial dysfunction.

3.3.4.3 Analysis

Flow mediated dilation is calculated as the percentage change in brachial artery

diameter in response to hyperaemia (equation 3.4). Baseline vessel diameter is taken as the average diameter from the first 2 minutes of brachial artery ultrasound imaging and the vessel diameter following hyperaemia is taken as the maximum diameter reached in the 2 minutes following cuff deflation.

ܨܯܦ = ܸܦ ℎݕ݌݁ݎܽ݁݉݅ܽ − ܤܽݏ݈݁݅݊݁ ܸܦ

ܤܽݏ݈݁݅݊݁ ܸܦ ݔ 100

FMD = Flow mediated dilation VD = vessel diameter

Equation 3.4 FMD dilation response

Previous studies have suggested that normal values for FMD should be considered to be between a 5-15% increase in brachial artery diameter and a 5-6 fold increase in blood flow on release of the BP cuff. A dilation response of between 0-5% indicates an impaired flow mediated dilation response 703, 704.

Nitroglycerin mediated dilation is calculated in the same way but as the percentage change in brachial artery diameter in response to nitroglycerin (equation 3.5).

ܰܯܦ = ܸܦ ݊݅ݐݎ݋݈݃ݕܿ݁ݎ݅݊݁ − ܤܽݏ݈݁݅݊݁ ܸܦ

ܤܽݏ݈݁݅݊݁ ܸܦ ݔ 100

NMD = Nitroglycerine mediated dilation VD = vessel diameter

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3.3.4.4 Circulating markers – von Willebrand factor

vWF levels were determined in this thesis via the analysis of fasting venous blood samples obtained by the author. The analysis was conducted by an experienced lab technician (Dr Lu Qin) according to the methodology outlined below:

• Following the collection of fasted venous blood samples into citrate tubes, the samples were centrifuged at 3000rpm for 15 minutes and the supernatant was aliqouted and stored in a -800C freezer.

• The citrated plasma was then thawed and analysed for vWf levels using a

standardised ELISA kit which was optomised according to previously established methods 705, 706 and conducted as follows:

1. A microtitre plate was coated with 100µl of dilated primary antiserum solution (30µl in 20.5ml coating buffer at pH 9.6) at room temperature and then refrigerated for a minimum of 60 minutes to over night 2. The microplate was washed 4 times with 250µl of wash buffer per well

before 100 µl of substrate was added with working strength detection antibody dilutant and incubated for 60 minutes at room temperature 3. The microplate was then washed 3 times with wash buffer before 100µl

of secondary antiserum was added and incubated at room temperature for 45 minutes

4. The microplate was then washed again for a final 3 times and 100µl of substrate was added and then incubated at room temperature for 20 minutes

5. The enzymatic reaction was then stopped by adding 50µl of hydrosulphuric acid

6. The absorbance of the solution was then immediately read on a microwell plate reader set at 492nm

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3.3.4.5 Ambulatory blood pressure monitoring (ABPM)

ABPM was the technique of choice for the assessment of systemic BP in this thesis and was conducted using the Cardiotens-01 device which is a computer operated

ambulatory BP and ECG monitor (Cardiotens-01, PMS Instruments, Maidenhead, UK). This device has been validated in accordance with recommended protocols and has been widely used in previous clinical studies 707-710. Its set up is such that a blood pressure cuff is positioned around the upper left arm and connected to a personal monitoring device worn around the waist, via a fibre optic cable. The device was programmed prior to use using the BP monitoring software Cardiovisions 1.7.2 (PMS Instruments, Maidenhead, UK) and customised to each individual, with regard to sleep and wake times and measurement intervals, amongst other factors. Measurements of BP are obtained automatically using an oscillometric method and in the event of a faulty reading the device is programmed to re-inflate a second time in order to avoid missed data points. For the purposes of this thesis the device was programmed to take readings at intervals of 15 minutes during daytime and intervals of 30 minute nocturnally. Patients were advised to carry out their normal daily activities and to complete a diary outlining any periods of unusual exertion or changes in activity for consideration when evaluating their BP variations. After the 24 hour period the BP data was downloaded and analysed using the ‘Medibase’ software program (Meditech, version 1.42).Maximum and

minimum, diurnal and nocturnal, SBP, DBP and MABP were recorded and the mean nocturnal dip in BP calculated (equation 3.6). Furthermore the short-term variability in SBP was determined for both the diurnal and nocturnal periods through calculation of the average coefficient of variation for each group (equation 3.7).

ܰ݋ܿݐݑݎ݈݊ܽ ܤܲ ݀݅݌ = ሺܦ݅ݑݎ݈݊ܽ ܯܣܤܲ − ܰ݋ܿݐݑݎ݈݊ܽ ܯܣܤܲሻ × 100

Where: MABP = mean arterial blood pressure, calculated according to equation 1.2

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ܥ݋݂݂݁݅ܿ݅݁݊ݐ ݋݂ ݒܽݎ݅ܽݐ݅݋݊ =ܵݐܽ݊݀ܽݎ݀ ݀݁ݒ݅ܽݐ݅݋݊

ܯ݁ܽ݊ ݔ 100

Equation 3.7 Coefficient of variation

3.3.4.6 Autonomic Nervous system assessment – Heart rate variability

Assessment of ANS function commonly falls into two categories, the first being the evaluation of BP and HR responses to provocative stimuli designed to test baroreflex sensitivity and the second being the analysis of systemic BP, resting HR and HRV over a 24 hour period 399. For the purposes of this thesis ANS function was assessed using 24 hour ECG monitoring and a frequency domain analysis of HRV using the Cardiotens- 01 device and ‘Medibase’ software introduced in the previous section (3.3.4.5). This device records continuous real time beat-to-beat ECG analysis via two independent channels following the precise placement of electrodes on the patient’s chest in the positions illustrated in figure 3.9. The electrodes are connected via a fibre optic cable to the personal monitoring device worn around the patient’s waist.

Figure 3.9 Diagrammatic representation of the electrode positioning for 24 hour ECG recording with the Cardiotens-01 device

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Following 24 hours of ECG recordings the data can be downloaded from the device and a full frequency domain HRV analysis performed by the ‘Medibase’ software using a series of validated algorithms. An assessment of HRV and ANS function can then be made through analysis of the LF and HF values and the LF/HF ratio (see section 1.7.2), recorded during the diurnal (active) phase, nocturnal (passive) phase and over the entire 24 hour period.